Project Summary/Abstract The development of highly isoform-selective poly(ADP-ribose) polymerase (PARP) biochemical probes will fill crucial knowledge gaps that exist in our understanding of biological functions of PARP enzymes. PARPs are a family of nuclear enzymes that catalyze poly(ADP-ribosyl)ation (PARylation) of substrate proteins such as histones. PARylation facilitates recruitment of DNA repair proteins. Consequently, PARP inhibitors (PARPi) are developed as a novel class of anticancer drugs that are used as single agents to treat BRCA-deficient tumors and as combination therapy with DNA damaging agents. The PARP superfamily is comprised of 17 members. Clinical PARPi are often associated with promiscuous inhibition of both PARP-1 and PARP-2 and some of them even inhibit other PARPs. This becomes a potential cause for their off-target hematologic toxicity. Due to non- specific targeting of multiple PARP-isoforms by currently known PARPi, a thorough interpretation of their pharmacological/clinical profiles has become highly complex. Further it was shown that PARP-1 inhibition alone is sufficient to repress the growth of MDA-MB-436 tumor xenograft. Depletion of both PARP-1 and PARP-2 led to embryonic lethality and aggressive T-cell lymphomas. One of the challenges impeding evaluation of PARP- isoform specific molecular interaction landscape in cellular context is the lack of potent and highly PARP-isoform selective chemical probes. Therefore, development of highly isoform selective PARP inhibitory probes is urgently needed. Weplan to address this by developing isoform-selective and potentially non-toxic novel PARP inhibitory chemical probes that will allow interrogation of changes in downstream signaling events of individual PARPs at a molecular level. The probes generated herein will also serve as template for the next generation preclinical agents. We will apply highly potent and novel set of UTT-lead compounds to develop exquisitely PARP-isoform selective chemical probes that will facilitate our understanding of biological functions of individual PARPs in both normal and diseased cells. As a proof-of-concept, we have synthesized and characterized several PARPi with nanomolar potency against both PARP-1 and PARP-2, with ~30-fold higher preference toward PARP-2. Based on this scientific premise and preliminary data, we propose the following specific aims: (A) to identify PARP-1 and PARP-2 selective biochemical probes; (B) to conduct in vitro PARP enzyme assays and isoform selectivity screening, and (C) to evaluate cytotoxicity of an isoform selective best PARPi in CAPAN-1 (BRCA2-/- and BRCA2cor) and SUM149 (BRCA1-/- and BRCA1cor) cells. These studies are expected to expand our knowledge on the impact of inhibiting a specific PARP-isoform by developed PARP chemical probes. By the end of the grant project period, we will be poised to evaluate cell-active and highly PARP-selective chemical probes in high value cell-based experiments. Subsequently, we plan to investigate PARP-isoform specific downstream signaling events, identification of new PARylated substrate proteins, on-target engagement and phenotypic change, and to validate if other PARPs are viable pharmacological targets.